Abstract
This protocol is designed to obtain base-resolution information on the level of 5-hydroxymethylcytosine (5hmC) in CpGs without the need for bisulfite modification. It relies on (i) the capture of hydroxymethylated sequences by a procedure known as ‘selective chemical labeling’ (see Szulwach et al., 2012) and (ii) the digestion of the captured DNA by exonucleases. After Illumina sequencing of the digested DNA fragments, an ad hoc bioinformatic pipeline extracts the information for further downstream analysis.
Keywords: 5-Hydroxymethylcytosine, Selective chemical labeling, Exonuclease digestion, CpG
Background
The methylation of cytosine in genomic DNA can be read by proteins and is mainly translated into gene silencing. Most CpG dinucleotides in the genome are methylated, including those located in gene regulatory regions such as enhancers. However, when required, these CpGs can be demethylated through oxidation of the methyl group by Ten Eleven Translocation (TET) enzymes and replacement by unmethylated cytosines by the base excision repair system. 5-Hydroxymethylcytosine (5hmC) is the first oxidative derivative of 5-methylcytosine, and mapping this modified base in the genome provides information on the regions undergoing active demethylation. Although selective chemical labeling (SCL) allows very specific detection of 5hmC, the resolution of this technique is limited by the size of the DNA fragments, especially when several CpGs are present in the captured DNA. In order to improve resolution, we have introduced a digestion step using exonucleases which trim the DNA molecule up to close proximity of the hydroxymethylated cytosines (Sérandour et al., 2016). Appropriate bioinformatic treatment of the sequencing reads then assigns hydroxymethylation score to the captured CpGs.
Materials and Reagents
Equipment
Procedure
Genomic DNA is extracted using the QIAGEN DNeasy kit and fragmented into 300 bp fragments by sonication. The enzyme β-glucosyltransferase catalyzes the addition of azide-glucose to 5hmCs present in the gDNA fragments. Azide then reacts with a biotin conjugate allowing immobilization of the modified DNA on streptavidin-coated magnetic beads (Figure 1A). After end repair, Illumina P7 adapter ligation and nick repair, the captured DNA is incubated with the 5’ → 3’ exonucleases lambda and RecJf. The lambda exonuclease digests one strand of the double-stranded DNA and stops when it encounters bead-bound biotinylated 5hmC, whereas the RecJf exonuclease digests single-stranded DNA that might result from digestion of unmodified contaminant DNA by the lambda exonuclease. After elution from the beads, the DNA is denatured into single-stranded DNA molecules. This is followed by second strand synthesis, ligation of the Illumina P5 adapter, PCR amplification and Illumina sequencing. Single end sequencing starts from the P5 adapter and identifies the location where the lambda exonuclease stopped digesting and its associated nearest hydroxymethylated CpG (Figure 1B). Figure 1. Overview of the SCL-exo procedure. A. As a first step of gDNA chemical modification, β-glucosyltransferase catalyzes the transfer of azide-glucose from UDP-6-N3-Glc to 5hmCs. Click chemistry is then used to add a biotin conjugate (DBCO-PEG4-Biotin) to the N3-Glc-modified 5hmCs. B. Flow chart of the SCL-exo protocol.
Data analysis
Information about data processing and analysis can be found in the original research article at: https://doi.org/10.1186/s13059-016-0919-y.
Notes
Recipes
Acknowledgments
We thank M. Bizot and G. Palierne for technical assistance. This work was funded by La Ligue Contre le Cancer, Cancéropole Grand Ouest, The CNRS and the University of Rennes 1. The authors declare no competing interests.
References
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